Methods for the synthesis of lactic acid using crabtree-negative yeast transformed with the lactate dehydrogenase gene

a technology of lactate dehydrogenase and crabtree-negative yeast, which is applied in the field of methods and materials involved in the production of organic products, can solve the problems of limited use of living bacteria as factories, difficult and expensive, and reduced pyruvate decarboxylase activity or alcohol dehydrogenase activity of cells, so as to achieve rapid growth and produce an organic product efficiently, and ph is neutral. , the effect of good synthesis

Inactive Publication Date: 2010-04-20
CARGILL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0142]A significant advantage of the present invention is that the preferred microorganisms, especially when grown under aerobic conditions, can utilize minimal media. The anaerobic production typically will not require additional nutrients, so the final product can be isolated from a relatively clean fermentation broth using any of a variety of separation techniques. Liquid-liquid extraction is a well known technique for the separation of organic acids from fermentation broths, and results in considerable purification. With the present invention it is believed that simpler, less costly, less energy-consuming systems may also be useful.

Problems solved by technology

The use of living bacteria as factories, however, is limited by the inability of the bacteria to grow as the organic product accumulates in the growth media.
Specifically, the ability of a microorganism to tolerate low pH obviates the need to maintain a neutral pH environment, which can be difficult and expensive during large-scale production processes.
The method can result in cells having reduced pyruvate decarboxylase activity or reduced alcohol dehydrogenase activity.

Method used

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  • Methods for the synthesis of lactic acid using crabtree-negative yeast transformed with the lactate dehydrogenase gene
  • Methods for the synthesis of lactic acid using crabtree-negative yeast transformed with the lactate dehydrogenase gene
  • Methods for the synthesis of lactic acid using crabtree-negative yeast transformed with the lactate dehydrogenase gene

Examples

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example 1

Recombinant Plasmid pHES / pSEH

[0147]0.5 μg of plasmid pGAD424 described by Chien et al. (Proc. Natl. Acad. Sci., 88(21):9578-9582 (1991)) was digested with the restriction enzyme HindIII. The digested mixture was separated by gel electrophoresis on a 0.8% agarose gel using TBE buffer. A 5.9 kbp fragment was then purified from the gel as described in Sambrook et al., (Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (1989)) . A complementary pair of 92 bp synthetic oligomers with multiple restriction enzyme recognition sites was designed. The first was designated fwd hes oligo and has the sequence identified as SEQ ID NO: 1. The second was designated camp hes oligo and has the sequence identified as SEQ ID NO: 2. 500 nmoles of the two complementary oligomers were annealed to each other by boiling for ten minutes and cooling gradually to room temperature. The double stranded 92 bp DNA was digested with HindIII and ligated to the HindIII digested 5.9 kbp...

example 2

PCR Amplification of Nucleic Acid Encoding Lactate Dehydrogenase from Lactobacillus helveticus and Pediococcus acidilactici

[0148]Genomic DNA was isolated from overnight cultures of Lactobacillus helveticus (ATCC 10797) and Pediococcus acidilactici (ATCC 25741) using PUREGENE® genomic DNA isolation kit (Gentra systems, Minneapolis, Minn.). PCR primers were designed to isolate lactate dehydrogenase-encoding nucleic acid from L. helveticus (lh-ldh oligos) and P. acidilactici (pa-ldh oligos) genomic DNA. These primers were designed based on the available gene sequences for lactate dehydrogenases in the Genbank databases, and have the sequences identified as SEQ ID NO 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The primers were optimized using Primer Designer software obtained from Sci-ed software (Durham, N.C.). One μmole of the genomic DNA was used along with 100 nmoles of primers. Pfu DNA polymerase (New England Biolabs) was used to PCR amplify lactate dehydrogenase (LDH) nucleic...

example 3

Cloning of L. helveticus and P. acidilactici into pCRII vector

[0155]PCR amplified LDH DNA products were ligated with pCRII vectors (FIGS. 3 and 4) using the TA cloning kit obtained from Invitrogen (Carlsbad, Calif.). The ligation mixture was then used to transform E. coli DR10B using methods described in Sambrook et al. (Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (1989)). The pCRII vectors supplied with the kit allowed for quick cloning of the PCR products according the manufacture's instructions. The pCRII vectors with the LDH genes from L. helveticus and P. acidilactici is depicted in FIG. 4.

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Abstract

The invention provides methods and materials related to the production of lactic acid. Specifically, the invention provides methods for producing lactic acid using a crabtree-negative yeast, such as of the Kluyveromyces, Pichia, Candida, Trichosporon and Yamadazmya genera, which have been transformed with a lactate dehydrogenase gene.

Description

[0001]This application is a continuation of U.S. patent application Ser. No. 10 / 287,564, entitled METHODS FOR THE SYNTHESIS OF LACTIC ACID USING CRABTREE-NEGATIVE YEAST TRANSFORMED WITH THE LACTATE DEHYDROGENASE GENE filed 4 Nov. 2002, now U.S. Pat. No. 7,229,805, which is a continuation of U.S. patent application Ser. No. 09 / 574,873, entitled ‘PRODUCTION OF LACTATE USING CRABTREE NEGATIVE ORGANISMS IN VARYING CULTURE CONDITIONS”, filed May 19, 2000, now U.S. Pat. No. 6,485,947, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 316,490, entitled “METHODS AND MATERIALS FOR THE SYNTHESIS OF ORGANIC PRODUCTS”, filed May 21, 1999, now abandoned the disclosure of which is hereby incorporated in its entirety herein.BACKGROUND[0002]1. Technical Field[0003]The invention relates to methods and materials involved in the production of organic products.[0004]2. Background Information[0005]Organic products such as lactic acid have many important industrial uses. For example,...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12P7/60C12N1/16C12N15/09C12P7/40C12P7/48C12P7/56C12R1/645
CPCC12N1/16C12P7/40C12P7/60C12P7/56C12P7/48Y02E50/10
Inventor RAJGARHIA, VINEETHATZIMANIKATIS, VASSILYOLSON, STACEYCARLSON, TINGSTARR, JOHN N.KOLSTAD, JEFFREY J.EYAL, AHARON
Owner CARGILL INC
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